Chronic fatigue syndrome (CFS) is characterized by fatigue, malaise, sleep and autonomic disturbances; it is considered a neuroimmune disorder with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, precipitated by stress and associated with high disability. CFS often occurs with comorbid diseases such as fibromyalgia, irritable bowel syndrome (IBS), interstitial cystitis (IC) and migraines, all of which also worsen by stress. There are no reliable animal models for CFS. Mast cells have emerged as a major regulator of neuroimmune endocrine processes affected by stress and have been implicated in all comorbid diseases associated with CFS. We have shown that: (a) mast cells have functional associations with nerve endings; (b) acute stress activates mast cells, an action blocked by pretreatment with corticotropin-releasing hormone (CRH) neutralizing antiserum, (c) stress increases blood-brain-barrier (BBB) permeability, which is inhibited by the CRH-receptor-1 (CRH-R1) antagonist Antalarmin and does not develop in mast cell deficient W/W mice, (d) human mast cells express CRH receptors, activation of which leads to selective release of vascular endothelial growth factor (VEGF), (e) some of the stimulatory effects of CRH on mast cells are mediated by neurotensin (NT), which has been shown to regulate the HPA axis. Tricyclic antidepressants are helpful in CFS and in the other comorbid diseases, but this mechanism of action is unknown. Our preliminary results show that the tricyclic antidepressant amitriptyline can inhibit rat mast cell secretion and intracellular calcium ion levels. Hypothesis: CRH, or the structurally related urocortin (Ucn), secreted by stress activates diencephalic mast cells, either alone or together with other neuropeptides such as NT leading to release of molecules that contribute to the central pathogenesis of CFS, and secretion of which can be inhibited by tricyclic antidepressants. We will investigate: Aim 1. The dose-response (0.1-100 uM) and time-course (0.5,1,6, 24 h) effects of three different classes of antidepressants, (a) the tricyclic (amitriptyline, imipramine), (b) the selective serotonin uptake inhibitors (fluoxetine, sertraline) and (c) bupropion on secretion of histamine, IL-1, IL-6, IL-8, IL-13, TNF, tryptase and VEGF from normal human umbilical cord-derived cultured mast cells (hCBMCs) derived from CD34+ progenitors triggered by IL-1, CRH or Ucn (100 nM). Aim 2. The effect of those antidepressants shown to be effective in Aim 1 for their ability to inhibit "brain mast cells" developed by culturing human umbilical cord matrix stem cells (hCMSCs) that are CD34- in the presence of 10 nM IL-4 and nerve growth factor (NGF), stimulated as in Aim 1 +/- NT (0.1-100 mM). Results from these studies will further our understanding of molecules released in response to stress hormones and which antidepressants may be useful in inhibiting these effects. Future studies will build on these findings to develop in vitro and in vivo models of CFS and lead to clinical trials with select antidepressants or other molecules that inhibit brain mast cells. [unreadable] [unreadable] [unreadable]